In the US, hepatitis C virus (HCV) is the leading cause of progressive hepatitis, cirrhosis and liver cell cancer. Understanding the type of immunity that allows approximately 15% people to clear acute hepatitis C holds the key to developing a vaccine. Exploratory studies in chimpanzee and humans show that T cells targeting multiple HCV proteins seem necessary for HCV clearance. To better understand the type of immunity that correlates with protection, careful study of successful responses will be required. Injection drug users have the highest rates of new HCV infection but typically many get lost to follow-up. In a local epidemiological cohort of over 200 injectors a high rate (28%/person/year) of new HCV infections has been identified and followed up. This now permits prospective study of people with acute hepatitis C. In this project, Aim 1 proposes to prospectively examine HCV-specific antibody and T cell responses from the point of diagnosis (based on conversion to HCV RNA or antibody positivity). The HCV protein targets and vigor of CD4+ T cell and CD8+ T cell responses will be determined by combining lymphoproliferation, intracellular cytokine staining, and ELISpot assays. Qualitative and quantitative antibody analysis will be conducted and CTL specific for HCV will be expanded, their repertoires examined and their epitopes and HLA restriction precisely mapped. MHC class I tetramers of some HLA/peptide epitopes will be synthesized for higher resolution comparison of CTL repertoires in resolvers and progressors. Evolution within peptide epitopes is hypothesized to result in viral escape from CTL. This model of immune escape will be tested in Aim 2, as CTL epitope variants are sequenced, synthesized and tested in CTL assays. An epitope whose ange of variants can still be recognized by CTL would constitute a logical vaccine component. Recent experiments have demonstrated expression of arrays of inhibitory and activating receptors (NKR) by HCV-specific CTL. The NKR ligand repertoire depends on the person's HLA type suggesting that NKR+ CTL may be variably regulated depending on ligand interaction in the liver. This will be tested in Aim 3. Overall this investigation will provide unique insights into protective mechanisms of immunity against HCV. It will also lay a foundation for vaccine design.